自噬逃脱线粒体DNA介导TLRs-MyD88-NFκB信号通路在呼吸机相关性肺损伤中的作用机制

It is demonstrated in our previous studies that TLR9, MyD88, NF-κB, IL-1β, IL-6 up-regulate significantly in the experimental VILI, but the roles of TLR ligands in the VILI have not yet been fully elucidated. Several studies have convincingly shown that neutrophil degranulation accumulation recruit by activated TLR9 via mitochondrial DNA after cell damage result in inflammatory response, contribute to organ damage. We sought to propose the hypothesis that, pressure tension will cause mitochondria damage and the escape of mitochondrial DNA thus mediate signal transduction which leads to inflammatory response, and eventually VILI. In order to elucidate the assumption, lung tissue and cells of mtDNA and DNase Ⅱ expressions were detected in models of VILI to clarify the mechanism of cell autophagy in VILI. And to investigate the source of mtDNA in the VILI, pulmonary macrophages, neutrophils, alveolar epithelial cells were extracted and detection of their mtDNA were required. Pulmonary macrophage in vivo and vitro were stimulated with agonists and inhibitor of TLR9, autophagy inducers and autophagy inhibitors respectively to detect the expressions of mtDNA, inflammatory cytokines and TLRs , and to investigate the relation between mtDNA and TLRs/MyD88/NF-κB signaling pathway which mediated inflammatory response. This research will further clarify the molecular mechanism of VILI, which would provide new clues of treatment for VILI.

前期研究表明：在VILI中TLR9、MyD88、NF-κB及炎性因子表达上调，但TLRs在VILI中的作用机制尚未清楚。有报道认为细胞受刺激后，线粒体DNA活化TLR9，募集趋化嗜中性粒细胞脱颗粒，造成器官损伤。我们假设：压力牵张导致细胞线粒体损伤，逃逸线粒体DNA介导信号传导，诱发炎症反应，导致VILI。为了阐明假设，建立VILI动物模型，研究mtDNA及DNaseⅡ的表达，阐明细胞自噬与VILI的作用机制；提取肺巨噬细胞、中性粒细胞、肺泡上皮细胞，检测mtDNA的表达，分析mtDNA的细胞来源；应用TLR9的激动剂、抑制剂及应用慢病毒转染、自噬诱导剂和自噬抑制剂分别作用于动物体内及体外肺巨噬细胞，研究mtDNA与炎性因子释放及TLRs表达，探讨mtDNA与TLRs/MyD88/NF-κB信号通路介导炎症反应的相关性,阐明VILI发生的分子机制，为有效防治VILI提供新的理论依据。

目的：探讨自噬逃脱线粒体DNA介导的TLR9/MyD88/NF-κB信号通路在VILI中的作用机制。.方法：对不同潮气量和通气时间的机械通气大鼠模型行WB检测自噬蛋白表达，透射电镜观察自噬小体数量，并检测ATP、ROS和ΔΨm变化。建立大鼠VILI模型后收集血清、BALF和肺组织。测定肺组织湿/干比（W/D），观察肺组织病理学和超微结构改变，ELISA测定血清和BALF炎症因子浓度，RT-qPCR和WB检测LC3B、COX IV和NF-κB mRNA及蛋白表达。采用AmA和CsA分别增强和抑制线粒体自噬，比较各组间肺水肿、肺损伤及炎症反应，检测DNase-II、mtDNA和LC3B表达，免疫荧光分析mtDNA来源。采用ODN2088和CQ分别抑制TLR9和mtDNA，比较各组间肺水肿、肺损伤及炎症反应，并检测COX IV、TLR9、MyD88和NF-κB p65 mRNA和蛋白表达。建立A549细胞牵张损伤模型，干扰或过表达PINK1、ODN2088和CQ分别抑制TLR9和mtDNA，探讨mtDNA介导TLR9/MyD88/NF-κB信号通路在细胞牵拉损伤中的作用，自噬慢病毒载体Stub-RFP-Sens-GFP-LC3B转染A549细胞后利用荧光显微镜下对比观察自噬流变化。.结果：.1. VT超过30 mL/kg、通气大于2.0 h可引起线粒体自噬，并ATP合成障碍、ROS积累和ΔΨm下降。大VT机械通气可明显引起急性肺组织炎症性损伤，且肺组织水肿、损伤和炎症反应程度与自噬程度与呈正相关。增强自噬时，血清DNase-II表达下调，胞内mtDNA 增加，肺组织炎症反应加重。.2. CON组和NTV组大鼠肺组织结构基本正常，HTV组肺组织见明显炎症性改变。与NTV组和CON组比较，HTV组大鼠肺W/D比、BALF总蛋白、血清炎性因子含量均明显升高， COX-4、TLR9、MyD88和NF-κB p65蛋白表达上调。ODN2088明显降低肺组织TLR9、MyD88、NF-κB p65表达；CQ明显降低肺组织COX-4、TLR9、MyD88、NF-κB p65表达；均能减轻大VT通气所致的炎性反应。.3.与CON组和5％CS组相比，20％CS组炎性因子含量显著增加。与CON组、20％CS组、Vector组和PINK1 siRNA组相比，PINK1 cDNA组Parkin、